The present invention relates to hybrid cell lines capable of continuously producing monoclonal antibody specific for neurotransmitter degrading enzymes and to immunoassay methods using the monoclonal antibodies.
In recent years, the capability to produce monoclonal antibodies specific to immunogenic determinants of bacterial cells, viruses, tissue components and proteins has provided a new spectrum of diagnostic and immunotherapeutic agents.
The major intracellular enzyme responsible for the metabolic degradation of catecholamines in mammals is monoamine oxidase (MAO). This enzyme, which is located in the outer mitochondrial membrane and exists in two forms (A and B), plays an important role in the nervous system. It is also believed to function in regulating the level of pressor amines, such as phenylethylamine, in the circulation and in non-neuronal tissues. MAO catalyzes the oxidation of amines to their corresponding aldehydes, which are rapidly metabolized, usually by oxidation to an acid.
Because MAO contributes to determining the concentration of the monoamine neurotransmitters, the activity of this enzyme has been studied in patients with a wide variety of neurological and psychiatric disorders. The B form of the enzyme is found in most human tissues (with the exception of placenta, where MAO A is the predominant form) and its presence in blood platelets provides a convenient tissue for study of its activity in pathological states. In fact, platelet MAO B level has been found to be reduced in chronic schizophrenic patients compared to normals in many studies, a finding which has led investigators to focus on MAO as a possible biological marker in schizophrenia. Low platelet MAO activity, however, has also been reported in bipolar affective illness, alcoholism, Down's Syndrome, iron deficiency anemia, essential hypertension, migraine, and juvenile diabetes. In addition, low MAO activity has been associated with suicide, sensation seeking and more frequent psychiatric counseling.
As indicated above there are two types of MAO, A and B, each of which has distinct catalytic properties and is expressed in various proportions in different tissues.
MAO A is selectively inhibited by low concentration of the irreversible active site inhibitor clorgyline and preferentially oxidizes low concentrations of 5-hydroxytryptamine.
MAO B is selectively inhibited by low concentrations of deprenyl and pargyline and preferentially oxidizes low concentrations of phenylethylamine, and benzylamine.
Although the distinguishing catalytic activities of MAO A and B have been evaluated to some degree, there is little known of the structure and molecular properties of MAO A and B. MAO B, the most extensively studied of the two enzymes, has been characterized as having molecular weight of approximately 120,000 and consists of two subunits of indistinguishable molecular weight. One subunit has an essential molecule of covalently bound flavin adenine dinucleotide. Moreover, MAO may have carbohydrate residues added to its polypeptides and its lipid microenvironment is thought to contribute significantly to its catalytic activity.
Since the measured level of MAO activity is a reflection of several factors (primary structure, covalent modification, and microenvironment), it is important to examine independently MAO activity and MAO concentration. Such a dissection of activity and concentration, along with sequencing information, should clarify the fundamental structure and function of this enzyme and suggest how alterations in them, if they exist, could lead to abnormal MAO activity in psychiatric disorders.
Several assays of MAO catalytic activity, including the most commonly used radioenzymatic assay, have been developed, but only one method has been available heretofore to measure the concentration of MAO protein.
The assay of active MAO concentration involves the titration of enzyme activity with known amounts of .sup.3 H-pargyline. This inhibitor reacts specifically and irreversibly on a mole to mole ratio with the covalently bound FAD of MAO B. Knowing the specific activity of the .sup.3 H-pargyline used in the reaction permits determination of the nmoles of pargyline bound in the preparation, which in turn is equal to the nmoles of MAO in the sample. Since the method measures only catalytically active MAO, molecules are not measured which are physiologically inactive or which are inactivated during extraction or non-specifically by the incubation conditions used for .sup.3 H-pargyline binding.
An alternative method for determining MAO concentrations involves the use of an antibody which binds MAO B specifically. Heretofore, the reagent antibody has been an antiserum prepared from rabbits immunized with highly purified MAO B. Although this reagent was used in rocket immunoelectrophoresis to determine the concentration of MAO in platelet samples from patients, the antiserum has had limited application because of variability in animal response to the MAO antigen and the relatively low titers obtained. Such difficulties are commonly encountered in producing specific antisera.
Accordingly, in efforts to determine accurately the molecular concentration of MAO or other neurotransmitter degrading enzymes, there exists a need for unlimited quantities of an unvarying antibody reagent which recognizes a single antigenic determinant of the enzymes. Such antibody reagents are provided by this invention.